**2. Cancer immunity and immunosuppression**

which has been tried to be triggered with a variety of strategies, such as immune checkpoint inhibitor- and engineered T cell-based therapies, to elicit antitumor immunity in the body and has shown great potential in treatment and prevention of recurrence of cancer, as exampled by recent striking outcomes of cancer immunotherapy in clinical applications [1, 2]. From the overall view, current cancer immunotherapy is usually undertaken in two ways: establishment of systemic immunity through utilizing cytokines, vaccines, or adoptive cell transfer (ACT) and regulation of local immunosuppressive tumor microenvironment through utilizing small molecules and immune checkpoint inhibitors. Immune checkpoint therapy addresses regulatory pathways in preexisting Ag-specific T cells aiming at enhancing antitumor immune responses, whereas self-sustaining systemic anticancer immunity proceeds with anticancer immune response, which is dictated by both vaccines and TME [3]. However, immune checkpoint inhibitors are mostly aimed at augmenting the potency of preexisting tumor-specific T cells and as such benefit only a portion of patients [2], while the strategies based on the engineered T cells involve complex bioengineering processes with almost an unacceptable cost and, sometimes, off-target severe toxicities [4]. These situations compel researchers to develop other anticancer tools, including, especially, the tumor Ag-containing vaccines, which trigger the immune system to establish anticancer immunity through several crucial processes: release of Ags from tumor beds to be taken up by Ag-presenting cells (APCs) or delivery of Ags to APCs, APC activation for presentation of tumor Ags, priming and activation of T cells by activated APCs, migration and infiltration of effector T cells back to the tumor, and finally the recognition and killing of tumor cells by effector T cells, each of which, theoretically, can be targeted with various therapeutic approaches [5]. In particular, cancer vaccines that are designed for targeting early steps of Ag processing are potentially able to enhance both therapeutic and prophylactic efficacies against not only primary tumor but also inoperable metastasis or relapse and will therefore benefit a wide range of patients, especially the ones that lack sufficient levels of preexisting tumor-specific T cells and immune

However, despite having in expectation tremendous therapeutic potential, cancer vaccines designed in conventional ways have been found elusive for successful treatment and eradication of tumors due to their weak immunogen insufficient to induce immune responses with conventional vaccination approaches. In addition, there are several other issues that may block the establishment of anticancer immunity, including degradation and rapid elimination of Ag, ineffective DCs uptake and Ag presentation, the suppression of T cell functions, and impairment of Ag presentation by the endoplasmic reticulum (ER) stress-driven lipid metabolism in DCs, thereby inhibiting protective T cell responses in cancer immunotherapy [7]. All of these highlight the need for developing new strategies to prepare cancer vaccines that can efficiently deliver tumor Ags and adjuvants to APCs and stimulate immune responses strong enough to kill tumor cells [8]. In this regard, NPs, such as liposomes, polymeric aggregates, and inorganic NPs, when used as a vaccine carrier have proven to be able to enhance the accumulation in draining lymph nodes (dLNs) of immunostimulants and adjuvant Ags, which thereby approach and stimulate a large number of Ag-presenting cells (APCs) enriched in dLNs to initiate cellular immunity required for fighting against cancer [6]. Moreover, several decades of intensive investigation on different types of NPs for targeting delivery

checkpoint-related molecules [6].

48 Immunization - Vaccine Adjuvant Delivery System and Strategies

Vertebrates are protected by the immune system from pathogens such as viruses, bacteria, fungi, and parasites through immune responses which can be classified into two categories, namely, innate and adaptive processes thus to establish, respectively, two types of immunity: the innate immunity providing rapid defense against pathogens and the more comprehensive adaptive immunity, which is set up requiring process of pathogens by professional APCs for presentation of immunogenic Ags to T and B cells to sponsoring cellular and humoral immune responses. Professional APCs, including B cells and macrophages, as well as dendritic cells (DCs) which are considered as the most efficient APC population, have proven to play a pivotal role at the interface of innate and adaptive immune responses [18, 19]. To initiate immune responses, DCs take up and then process endogenous or exogenous Ags in the context of major histocompatibility complex (MHC) class I or II, followed by presentation of the MHC-I or -II/Ag peptide complex as the activation "signal 1", respectively, to CD8+ and CD4+ T cells, which are activated requiring an additional "signal 2" induced by ligation of co-stimulatory markers CD80/86 on DCs with CD28 on T cells, as well as a T cell polarizing "signal 3" provided by cytokines, such as interleukins (ILs) and interferons (IFNs) secreted by DCs [6]. Although MHC-I is constitutively expressed by most mammalian cells, nonprofessional APCs can never present "signals 2 and 3" to alert the immune system after invasion by pathogens, highlighting the Ag processing and presentation by APCs as a critical first step in sponsoring adaptive immune responses. In particular, DCs rather than other APCs prove to be able to process exogenous pathogens to activate CD8+ T cells via a unique process called cross-presentation, of which, though the exact mechanisms are still unclear, the vacuolar and cytosolic pathways have been identified to utilize endosomes and endoplasmic reticulum, respectively, to generate the MHC-I/Ag peptide complexes [20–22]. Notably, the endosome pathway in normal conditions is devoted to process the exogenous Ags to form the MHC-II/ Ag peptide complexes for activation of CD4+ T cells, although the abnormal increase in endosomal pH or occurrence of endosome breakup made purposely by artificial strategies is thought to prevent the protease-mediated degradation of Ags in endosomes, thus promoting cross-presentation [12, 23], and additionally, certain DC subsets such as tissue-resident CD8+ and migratory CD103+ DCs in mice and CD141+/BDCA-3+ DCs in humans were reported to be more efficient at Ag cross-presentation than other DC subtypes [24, 25].

excessive activation of T cell responses, suggesting that development of vaccines for cancer immonotherapy is still confronting a huge challenge arising from cancer immunosuppression.

Vaccines Developed for Cancer Immunotherapy http://dx.doi.org/10.5772/intechopen.80889 51

Tumor Ags include mutated cell surface components, such as polysaccharides, peptides, oncoproteins, and DNA and mRNA that encode those proteins, which as referred to subunit Ags, meanwhile tumor cell lysate and immunogenically dying tumor cells can also serve as the source of whole-cell Ags [6]. As key components utilized for formulating anticancer vaccines, subunit Ags have major advantages including defined chemical synthesis; ease of production; and for vaccine formulations, requiring, possibly, no Ag-processing by APCs and challenges including elicitation of humoral rather than cellular immune responses, poor delivery efficiency, and in vivo stability. Whole-cell Ags have major advantages including broad-epitope immune responses, potential for "personalized" therapy, full preservation of tumor Ags and challenges including production requiring tissue biopsy, difficulty in manufacturing, loss of antigenicity during production, presence of self-Ags, and immunosuppressive molecules such as PD-L1. Notably some viruses, such as Epstein–Barr virus (EBV), human papilloma virus (HPV), and hepatitis B and C viruses, have proven to contribute to certain cancer-related development, and therefore, their virally gene encoded surface proteins may also serve as the potential target Ags to constitute the vaccines for cancer immunotherapy [35, 36]. Among different types of tumor Ags, oncoproteins, which are encoded by oncogenes involved in the regulation or synthesis of proteins linked to tumor cell growth and may also be either mutated or overexpressed normal or embryonic proteins from fetal development, are intensively investigated for cancer vaccines since they have a big potential in induction of broad-epitope CD8+ and CD4+ T cell responses. Notably, compared to full-length protein-based Ags that require cellular uptake and processing for presentation to T cells, peptide epitopes can directly bind to MHC molecules and thus directly activate T cells and, moreover, are more endurable to damages during the preparation and storage of vaccine products, thus, in line with these advantages, leading to many ongoing clinical trials on peptide-based cancer vaccines [37, 38]. However, poor immunogenicity and limited therapeutic efficacy are still big challenges in developing protein, especially, peptide Ag-based subunit vaccines that are designed for cancer immunotherapy; for example, in the case of melanoma, the identified Ags include β-catenin, survivin, tyrosinase, gp100, MAGE, melan-A (MART1), and NY-ESO-1, some of which, such as gp100 and MAGE-A3 peptides, when tested in clinical trials just showed only moderate or null therapeutic efficacy [39]. Grooming through clinical trials on peptide-based cancer vaccines, it may be safely concluded that therapeutic efficacy of subunit vaccines against cancer remains suboptimal [2], due to at least partially the fact that many tumor Ags evaluated in clinical trials are self-Ags which can hardly trigger the autoreactive T cells leading to immunotolerance [6]. These disappointed outcomes highlight that the conventional subunit vaccines should actually be formulated with innovative modalities, which may be an alternative promising strategy to further improve cancer immunotherapy, as evidenced by positive results obtained from pre- and clinical investigations carried out more recently

**3. NP entrapping various Ags for delivering cancer vaccines**

After activation and differentiation from CD8+ T cells in lymphoid tissues, the matured Ag-specific cytotoxic CD8+ T lymphocytes (CTLs) enter the systemic circulation and patrol peripheral tissues in search of target cells, which display a specific Ag epitope in the context of MHC-I matching the Ag-specific T cell receptors (TCRs) on CTLs, which once identification of the target cells will secrete perforin and granzymes to lyse them and within minutes move on to kill the next target [26]. By contrast, CD4+ T cells mainly play a helper role of regulation of immune responses as manifested by the observations that after activation by MHC-II/Ag peptide complex presented by DCs, naïve CD4+ T cells differentiate into four distinctive subtypes depending on the polarizing cytokines [27]. Type 1 helper T cells (Th1) induced by IL-12 secrete IL-2 and IFN-γ to promote CD8+ T cell responses; Th2 cells induced by IL-4 secrete IL-4 and IL-5 and are involved in humoral immune responses; regulatory T cells (Tregs) induced by IL-2 and TGF-β (transforming growth factor beta) secrete TGF-β and IL-10 to suppress immune responses; and Th17 cells induced by TGF-β, IL-6, and IL-21 secrete IL-17 and IL-22 to break immune tolerance and possibly leading to autoimmunity [27, 28]. In addition, it is reported that CD4+ helper T cells are utilizing the expressed CD40L for feeding back to DCs to further amplify immune activation and aid in establishment of memory CD8+ T cell responses [29, 30].

To prevent cancerous occurrence, the immune system constantly implements a process referred to as immunosurveillance whereby to inhibit oncogenesis by actively identifying and eliminating tumor cells, which however, have also devised mechanisms to evade immune responses, including downregulation of tumor Ags and promotion of immunosuppression [31, 32]. In established tumor microenvironment, it is generally immunosuppressive due to upregulation or production of inhibitory molecules, such as TGF-β1, CXCL12, VEGF, ARG1 (Arginase1), CCL18, iNOS (nitric oxide synthase), IL-10, IL-35, and galectin-1 by many types of cells, including cancer-associated fibroblasts, myeloid-derived suppressor cells, Tregs, and tumor-associated macrophages (TAMs), against T cells [33]. Also, activated T cells upregulate CTLA-4 (CTL-associated protein 4) which binds to co-stimulatory molecules on DCs with higher affinity than CD28, serves as a peripheral inhibitory signal to prevent over-reactivity of T cells, and dampens antitumor immune responses. Besides, tumor cells can also secrete cytokines such as IL-10 and TGF-β, which both directly inhibit the proliferation of CTLs and drive the differentiation of Tregs to provide an additional source of immunosuppressive cytokines, while subsets of tumor cells highly express programmed death-ligand 1 (PD-L1) for binding to programmed death-1 (PD-1) on T cells and inhibiting their effector functions [34]. Thus, tumor cells can promote immunosuppressive tumor microenvironment and shield themselves from CTLs by hijacking normal negative feedback loops designed to guard against excessive activation of T cell responses, suggesting that development of vaccines for cancer immonotherapy is still confronting a huge challenge arising from cancer immunosuppression.
